Proteins produced in cell culture can exhibit a high level of heterogeneity. Part of the heterogeneity is due to the glycosylation pattern of the protein (which includes the addition of galactose to certain amino acid residues of the protein). Glycosylation patterns are strongly influenced by cell culture conditions. Different types of glycosylation variants include G0 variants, G1 variants, G2 variants, and high-mannose variants. The variants for example, involve the addition of galactose to an asparagine residue within the amino acid sequence of the recombinant protein, such as in the Fc region of an antibody. The glycosylation (including galactosylation) profile of a particular protein can influence its biological activity due to variable effects on folding, stability, efficacy, and half-life. The glycosylation pattern of a particular protein can be determined by the use of various assays known in the art, such as assays for releasing oligosaccharides present on a particular protein via enzymatic digestion, analysing the variants by chromatography, or other methods in the art.
Glycoproteins are involved in immune defence, cell growth, and cell-cell adhesion, and the glycans that mediate these functions can take on a myriad of complex structures. More than 90% of therapeutic proteins in existence are glycoproteins. For instance, many different recombinant forms of immunoglobulins (e.g., monoclonal antibodies, mAbs) are produced as therapeutic glycoprotein drugs for treating serious conditions. These therapeutic proteins (or glycoproteins) contain complex oligosaccharide moieties whose presence, absence, and profile (i.e. relative proportion of variants present) can have significant impact on therapeutic efficacy, pharmacokinetics, immunogenicity, folding, and stability of the therapeutic protein. For example, certain glycan structures are known to cause aggregation and decrease efficacy of a therapeutic protein. The proportion and pattern of glycosylation variants that are present on a therapeutic protein are a result of the cell culture conditions used in the production of the therapeutic protein. Accordingly, throughout the process of protein production (e.g. fermentation, purification, and formulation) the types and proportions of glycosylation variants present within a population of therapeutic protein should be characterised.
A variety of approaches can be used to characterise glycoproteins and their particular glycan moieties. For example, high-performance liquid chromatography (HPLC) followed by quadrupole time-of-flight mass spectrometry (LC/QTOF MS) can distinguish the number of glycan (such as, galactose) units attached to an intact protein, thus providing the ability to distinguish an active form from an inactive one. In addition, glycosylation sites can be elucidated by digesting the particular glycoprotein with trypsin and using LC/QTOF MS to separate and identify the resulting glycopeptides. Comparison of the masses of these peptides to those generated by a theoretical digestion of the desired glycan form of the protein can determine if the protein is properly glycosylated. Capillary electrophoresis (CE) coupled to QTOF MS can also be used for this purpose.
Analysis of the glycan moieties attached to a protein can also be done by enzymatic deglycosylation and hydrolysis. N-Glycosidase F (PNGase F), an amidase, is used to cleave asparagine-linked (N-linked) oligosaccharides from glycoproteins. Most commonly, the removed glycans are derivatized, labelled, and analysed by fluorescence detection.
Unlabelled glycans can also be analysed by various techniques. Traditionally, high performance anion exchange chromatography with pulsed ampiometric detection (HPAEC-PAD) is used to separate unlabelled glycans based on hydroxyl group interaction with the stationary phase. HPAEC-PAD is also used to determine sialic acid content, which is measured to ensure the product is safe and for batch-to-batch reproducibility. While this method removes the potential issues with labelling efficiency, it still provides fingerprint-like information and requires standards for glycan identification. Gas chromatography (GC) is also frequently used for monosaccharide compositional analysis because it is robust and has high resolution.
In the case of producing a biosimilar, it is imperative that a glycosylation pattern is as close as possible as the reference protein in order that the pharmacokinetics of the biosimilar closely matches those of the reference protein. It is known in the art that the culture conditions and production methods can influence the glycosylation profile of a protein produced in a cell culture method. Various factors in cell culture are inextricably linked, such that merely adding for example, galactose to media may on the one hand favourably influence the galactosylation pattern of the protein but may, on the other hand also have other, negative effects on other characteristics of the protein, such as the charge profile, proportion of protein fragments, proportion of aggregates, and titre protein. Furthermore, cell viability may be affected.